Compositions and methods comprising male fertility sequences

ABSTRACT

Compositions and methods for modulating male fertility in a plant are provided. Compositions comprise nucleotide sequences, and active fragments and variants thereof, which modulate male fertility. Further provided are expression cassettes comprising the male fertility polynucleotides, or active fragments or variants thereof, operably linked to a promoter, wherein expression of the polynucleotides modulates the male fertility of a plant. Various methods are provided wherein the level and/or activity of the sequences that influence male fertility is modulated in a plant or plant part.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of patent application Ser. No.14/425,916, filed Mar. 4, 2015, now U.S. Pat. No. 10,155,962, issuedDec. 18, 2018, which is a 371 national stage entry of PCT patentapplication PCT/US13/058500, filed Sep. 6, 2013, which claims benefit ofand priority to Provisional Application No. 61/697,590, filed Sep. 6,2012, each of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of plant molecular biology,more particularly to influencing male fertility.

REFERENCE TO ELECTRONICALLY-SUBMITTED SEQUENCE LISTING

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named5282-PCT ST25.txt, last modified on Sep. 6, 2013, having a size of 42KB, and is filed concurrently with the specification. The sequencelisting contained in this ASCII formatted document is part of thespecification and is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Development of hybrid plant breeding has made possible considerableadvances in quality and quantity of crops produced. Increased yield andcombination of desirable characteristics, such as resistance to diseaseand insects, heat and drought tolerance, along with variations in plantcomposition are all possible because of hybridization procedures. Theseprocedures frequently rely heavily on providing for a male parentcontributing pollen to a female parent to produce the resulting hybrid.

Field crops are bred through techniques that take advantage of theplant's method of pollination. A plant is self-pollinated if pollen fromone flower is transferred to the same or another flower of the sameplant or a genetically identical plant. A plant is cross-pollinated ifthe pollen comes from a flower on a genetically different plant.

In certain species, such as Brassica campestris, the plant is normallyself-sterile and can only be cross-pollinated. In self-pollinatingspecies, such as soybeans and cotton, the male and female plants areanatomically juxtaposed. During natural pollination, the malereproductive organs of a given flower pollinate the female reproductiveorgans of the same flower.

Bread wheat (Triticum aestivum) is a hexaploid plant having three pairsof homologous chromosomes defining genomes A, B and D. The endosperm ofwheat grain comprises 2 haploid complements from a maternal cell and 1from a paternal cell. The embryo of wheat grain comprises one haploidcomplement from each of the maternal and paternal cells. Hexaploidy hasbeen considered a significant obstacle in researching and developinguseful variants of wheat. In fact, very little is known regarding howhomologous genes of wheat interact, how their expression is regulated,and how the different proteins produced by homologous genes functionseparately or in concert.

An essential aspect of much of the work underway with genetic malesterility systems is the identification of genes influencing malefertility. Such a gene can be used in a variety of systems to controlmale fertility including those described herein.

BRIEF SUMMARY OF THE INVENTION

Compositions and methods for modulating male fertility in a plant areprovided. Compositions comprise nucleotide sequences, and activefragments and variants thereof, which modulate male fertility. Furtherprovided are expression cassettes comprising one or more of the malefertility polynucleotides, or active fragments or variants thereof,operably linked to a promoter, wherein expression of the polynucleotidesmodulates the male fertility of a plant. Various methods are providedwherein the level and/or activity of a polynucleotide that influencesmale fertility is modulated in a plant or plant part.

DESCRIPTION OF THE FIGURES

FIG. 1 shows an alignment of the Wheat MS26 genes, A (SEQ ID NO: 28), B(SEQ ID NO: 29) and D (SEQ ID NO: 30) genomes, across the MS26+ targetsite (SEQ ID NO: 21) compared to the maize (SEQ ID NO: 31), sorghum (SEQID NO: 32) and rice (SEQ ID NO: 33) MS26 orthologous genes.

FIG. 2 shows an alignment of the NHEJ mutations induced by the MS26+homing endonuclease, described herein. The mutations were identified bydeep sequencing. The reference illustrates the unmodified locus with thegenomic target site underlined. The expected site of cleavage is alsoindicated. Deletions as a result of imperfect NHEJ are shown by a “-”.The reference corresponds to Fielder wheat Ms26 (SEQ ID NO: 34).

FIG. 3 shows types of NHEJ mutations induced by the MS26+ homingendonuclease, described herein. The mutations were identified bysequencing of subcloned PCR products in DNA vectors. MS26 alleledesignation 1, 2, and 3 likely refers to wheat genome copy D, A and Brespectively.

FIG. 4 shows an alignment of the NHEJ mutations induced by the MS26+homing endonuclease. The top sequence is the MS26 target site (SEQ IDNO: 21) compared to a reference sequence (SEQ ID NO: 45) whichillustrates the unmodified locus. Deletions as a result of imperfectNHEJ are shown by a “-”, while the gap represents a C nucleotideinsertion in SEQ ID NO: 50. The mutations were identified by sequencingof subcloned PCR products in DNA vectors.

DETAILED DESCRIPTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

I. Male Fertility Polynucleotides

Compositions disclosed herein include polynucleotides and polypeptidesthat influence male fertility. In particular, isolated polynucleotidesare provided comprising nucleotide sequences encoding the amino acidsequences set forth in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, or 18 oractive fragments or variants thereof. Further provided are polypeptideshaving an amino acid sequence encoded by a polynucleotide describedherein, for example those set forth in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13,15, or 17 or active fragments or variants thereof.

Sexually reproducing plants develop specialized tissues specific for theproduction of male and female gametes. Successful production of malegametes relies on proper formation of the male reproductive tissues. Thestamen, which embodies the male reproductive organ of plants, containsvarious cell types, including for example, the filament, anther,tapetum, and pollen. As used herein, “male tissue” refers to thespecialized tissue in a sexually reproducing plant that is responsiblefor production of the male gamete. Male tissues include, but are notlimited to, the stamen, filament, anther, tapetum, and pollen.

The process of mature pollen grain formation begins withmicrosporogenesis, wherein meiocytes are formed in the sporogenoustissue of the anther. Microgametogenesis follows, wherein microsporesdivide mitotically and develop into the microgametophyte, or pollengrains. The condition of “male fertility” or “male fertile” refers tothose plants producing a mature pollen grain capable of fertilizing afemale gamete to produce a subsequent generation of offspring. The term“influences male fertility” or “modulates male fertility”, as usedherein, refers to any increase or decrease in the ability of a plant toproduce a mature pollen grain when compared to an appropriate control. A“mature pollen grain” or “mature pollen” refers to any pollen graincapable of fertilizing a female gamete to produce a subsequentgeneration of offspring. Likewise, the term “male fertilitypolynucleotide” or “male fertility polypeptide” refers to apolynucleotide or polypeptide that modulates male fertility. A malefertility polynucleotide may, for example, encode a polypeptide thatparticipates in the process of microsporogenesis or microgametogenesis.

Male fertility polynucleotides disclosed herein include homologs andorthologs of polynucleotides shown to influence male fertility. Forexample, male fertility polynucleotides, and active fragments andvariants thereof, disclosed herein include homologs and orthologs ofMs22 (also referred to as Msca1). Mutagenesis studies of Ms22 resultedin phenotypically male sterile maize plants with anthers that did notextrude from the tassel and lacked sporogenous tissue. West andAlbertsen (1985) Maize Newsletter 59:87; Neuffer et al. (1977) Mutantsof maize. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

Plants deficient in Ms22 expression exhibit physiological changes earlyin reproductive-tissue development. Ms22 is believed to have a role inanother development that occurs earlier than that of Ms45 or Ms26.Certain male sterility genes such as MAC1, EMS1 or GNE2 (Sorensen et al.(2002) Plant J. 29:581-594) prevent cell growth in the quartet stage.Mutations in the SPOROCYTELESS/NOZZLE gene act early in development, butimpact both anther and ovule formation such that plants are male andfemale sterile. The SPOROCYTELESS gene of Arabidopsis is required forinitiation of sporogenesis and encodes a novel nuclear protein (GenesDev. 1999 Aug. 15; 13(16):2108-17). Because Ms22 is critical for theprogression of microsporogenesis, maintenance of male sterility in Ms22mutants is very reliable compared to other male sterility mutantconstructs. As disclosed elsewhere herein, Ms22 polynucleotides fromwheat are set forth in SEQ ID NOs: 1, 3, and 5.

Additional male fertility polynucleotides include the Ms26polynucleotide and homologs and orthologs thereof. Ms26 polypeptideshave been reported to have significant homology to P450 enzymes found inyeast, plants, and mammals. P450 enzymes have been widely studied andcharacteristic protein domains have been elucidated. The Ms26 proteincontains several structural motifs characteristic of eukaryotic P450's,including the heme-binding domain FxxGxRxCxG (domain D; SEQ ID NO: 19),domain A A/GGXD/ETT/S (dioxygen-binding; SEQ ID NO: 20), domain B(steroid-binding) and domain C. Phylogenetic tree analysis revealed thatMs26 is most closely related to P450s involved in fatty acidomega-hydroxylation found in Arabidopsis thaliana and Vicia saliva. See,for example, US Patent Publication No. 2012/0005792, herein incorporatedby reference. As disclosed elsewhere herein, Ms26 polynucleotides fromwheat are set forth in SEQ ID NOs: 7, 9, and 11.

Additional male fertility polynucleotides, and active fragments andvariants thereof, disclosed herein may also include homologs andorthologs of Ms45 polynucleotides. The Ms45 polynucleotide is a malefertility polynucleotide characterized in maize. Mutations of Ms45 canresult in breakdown of microsporogenesis during vacuolation of themicrospores rendering the mutated plants male sterile. When the clonedmaize Ms45 polynucleotide is introduced into such mutated male sterileplants, the gene can complement the mutation and confer male fertility.As disclosed elsewhere herein, Ms45 polynucleotides from wheat are setforth in SEQ ID NOs: 13, 15, and 17.

Strategies for manipulation of expression of male-fertilitypolynucleotides in wheat will require consideration of the ploidy levelof the individual wheat variety. Triticum aestivum is a hexaploidcontaining three genomes designated A, B, and D (N=21); each genomecomprises seven pairs of nonhomologous chromosomes. Einkorn wheatvarieties are diploids (N=7) and emmer wheat varieties are tetraploids(N=14).

Isolated or substantially purified nucleic acid molecules or proteincompositions are disclosed herein. An “isolated” or “purified” nucleicacid molecule, polynucleotide, or protein, or biologically activeportion thereof, is substantially or essentially free from componentsthat normally accompany or interact with the polynucleotide or proteinas found in its naturally occurring environment. Thus, an isolated orpurified polynucleotide or protein is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Optimally, an “isolated”polynucleotide is free of sequences (optimally protein encodingsequences) that naturally flank the polynucleotide (i.e., sequenceslocated at the 5′ and 3′ ends of the polynucleotide) in the genomic DNAof the organism from which the polynucleotide is derived. For example,in various embodiments, the isolated polynucleotide can contain lessthan about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotidesequence that naturally flank the polynucleotide in genomic DNA of thecell from which the polynucleotide is derived. A protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) ofcontaminating protein. When the polypeptides disclosed herein orbiologically active portion thereof is recombinantly produced, optimallyculture medium represents less than about 30%, 20%, 10%, 5%, or 1% (bydry weight) of chemical precursors or non-protein-of-interest chemicals.

A “subject plant” or “subject plant cell” is one in which geneticalteration, such as transformation, has been effected as to a gene ofinterest, or is a plant or plant cell which is descended from a plant orcell so altered and which comprises the alteration. A “control” or“control plant” or “control plant cell” provides a reference point formeasuring changes in phenotype of the subject plant or plant cell.

A control plant or plant cell may comprise, for example: (a) a wild-typeplant or plant cell, i.e., of the same genotype as the starting materialfor the genetic alteration which resulted in the subject plant or cell;(b) a plant or plant cell of the same genotype as the starting materialbut which has been transformed with a null construct (i.e. with aconstruct which has no known effect on the trait of interest, such as aconstruct comprising a marker gene); (c) a plant or plant cell which isa non-transformed segregant among progeny of a subject plant or plantcell; (d) a plant or plant cell genetically identical to the subjectplant or plant cell but which is not exposed to conditions or stimulithat would induce expression of the gene of interest; or (e) the subjectplant or plant cell itself, under conditions in which the gene ofinterest is not expressed.

A. Fragments and Variants of Male Fertility Sequences

Fragments and variants of the disclosed polynucleotides and proteinsencoded thereby are also provided. By “fragment” is intended a portionof the polynucleotide or a portion of the amino acid sequence and henceprotein encoded thereby. Fragments of a polynucleotide may encodeprotein fragments that retain the biological activity of the nativeprotein and hence influence male fertility. Alternatively, fragments ofa polynucleotide that are useful as hybridization probes generally donot encode fragment proteins retaining biological activity. Thus,fragments of a nucleotide sequence may range from at least about 20nucleotides, about 50 nucleotides, about 100 nucleotides, and up to thefull-length polynucleotide encoding the polypeptides disclosed herein.

A fragment of a polynucleotide that encodes a biologically activeportion of a polypeptide that influences male fertility will encode atleast 15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 525,or 537 contiguous amino acids, or up to the total number of amino acidspresent in a full-length polypeptide that influences male fertility (forexample, SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, and 18, respectively).Fragments of a polynucleotide encoding a polypeptide that influencesmale fertility that are useful as hybridization probes or PCR primersgenerally need not encode a biologically active portion of a polypeptidethat influences male fertility.

Thus, a fragment of a male fertility polynucleotide as disclosed hereinmay encode a biologically active portion of a male fertilitypolypeptide, or it may be a fragment that can be used as a hybridizationprobe or PCR primer using methods disclosed below. A biologically activeportion of a male fertility polypeptide can be prepared by isolating aportion of one of the male fertility polynucleotides disclosed herein,expressing the encoded portion of the male fertility protein (e.g., byrecombinant expression in vitro), and assessing the activity of theencoded portion of the male fertility polypeptide. Polynucleotides thatare fragments of a male fertility polynucleotide comprise at least 16,20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, or 1629nucleotides, or up to the number of nucleotides present in a full-lengthmale fertility polynucleotide disclosed herein (i.e., SEQ ID NOS:1, 3,5, 7, 9, 11, 13, 15, or 17, respectively).

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more internal sites within the nativepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the native polynucleotide. As used herein, a “native”or “wild type” polynucleotide or polypeptide comprises a naturallyoccurring nucleotide sequence or amino acid sequence, respectively. Forpolynucleotides, conservative variants include those sequences that,because of the degeneracy of the genetic code, encode the amino acidsequence of one of the male fertility polypeptides disclosed herein.Naturally occurring allelic variants such as these can be identifiedwith the use of well-known molecular biology techniques, as, forexample, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant polynucleotides also includesynthetically derived polynucleotides, such as those generated, forexample, by using site-directed mutagenesis but which still encode amale fertility polypeptide. Generally, variants of a particularpolynucleotides disclosed herein will have at least about 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to that particularpolynucleotide (e.g., any one of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15,or 17) as determined by sequence alignment programs and parametersdescribed elsewhere herein.

Variants of a particular polynucleotide disclosed herein (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Thus, for example, an isolated polynucleotide thatencodes a polypeptide with a given percent sequence identity to thepolypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18 aredisclosed. Percent sequence identity between any two polypeptides can becalculated using sequence alignment programs and parameters describedelsewhere herein. Where any given pair of polynucleotides disclosedherein is evaluated by comparison of the percent sequence identityshared by the two polypeptides they encode, the percent sequenceidentity between the two encoded polypeptides is at least about 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity.

“Variant” protein is intended to mean a protein derived from the nativeprotein by deletion or addition of one or more amino acids at one ormore internal sites in the native protein and/or substitution of one ormore amino acids at one or more sites in the native protein. Variantproteins disclosed herein are biologically active, that is they continueto possess the desired biological activity of the native protein, thatis, male fertility activity as described herein. Such variants mayresult from, for example, genetic polymorphism or from humanmanipulation. Biologically active variants of a male fertility proteindisclosed herein will have at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to the amino acid sequence for the native protein(e.g. any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18) asdetermined by sequence alignment programs and parameters describedelsewhere herein. A biologically active variant of a protein disclosedherein may differ from that protein by as few as 1-15 amino acidresidues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2,or even 1 amino acid residue.

The proteins disclosed herein may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants and fragments of the malefertility polypeptides can be prepared by mutations in the DNA. Methodsfor mutagenesis and polynucleotide alterations are well known in theart. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S.Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques inMolecular Biology (MacMillan Publishing Company, New York) and thereferences cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al. (1978) Atlas ofProtein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.), herein incorporated by reference. Conservative substitutions,such as exchanging one amino acid with another having similarproperties, may be optimal.

Thus, the genes and polynucleotides disclosed herein include both thenaturally occurring sequences as well as DNA sequence variants whichretain function. Likewise, the male fertility polypeptides and proteinsencompass both naturally occurring polypeptides as well as variationsand modified forms thereof. Such polynucleotide and polypeptide variantswill continue to possess the desired male fertility activity. Themutations that will be made in the DNA encoding the variant must notplace the sequence out of reading frame and optimally will not createcomplementary regions that could produce secondary mRNA structure. See,EP Patent Application Publication No. 75,444.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays. That is, the activity can beevaluated by assaying for male fertility activity.

Increases or decreases in male fertility can be assayed in a variety ofways. One of ordinary skill in the art can readily assess activity ofthe variant or fragment by introducing the polynucleotide into a planthomozygous for a stable male sterile allele of the polynucleotide, andobserving male tissue development in the plant. For example, to assayfor male fertility activity of Ms22 (i.e. SEQ ID NO: 1, 3, or 5), one ofskill in the art can begin by constructing a plant homozygous for amutation in the native Ms22 gene resulting in male sterility.Subsequently, one could complement the mutation by providing the Ms22polynucleotide, or active fragment or variant thereof, and observingwhether the male tissues of the plant develop normally and are able toproduce mature pollen. Likewise, the same procedure can be carried outto assay for the male fertility activity of variants or fragments ofMs26 (i.e. SEQ ID NO: 7, 9, or 11) or Ms45 (i.e. SEQ ID NO: 13, 15, or17), also disclosed herein.

Variant functional polynucleotides and proteins also encompass sequencesand proteins derived from a mutagenic and recombinogenic procedure suchas DNA shuffling. With such a procedure, one or more different malefertility sequences can be manipulated to create a new male fertilitypolypeptide possessing the desired properties. In this manner, librariesof recombinant polynucleotides are generated from a population ofrelated sequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between the male fertilitypolynucleotides disclosed herein and other known male fertilitypolynucleotides to obtain a new gene coding for a protein with animproved property of interest, such as an increased K_(m) in the case ofan enzyme. Strategies for such DNA shuffling are known in the art. See,for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech.15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al.(1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998)Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

II. Sequence Analysis

As used herein, “sequence identity” or “identity” in the context of twopolynucleotides or polypeptide sequences makes reference to the residuesin the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

The use of the term “polynucleotide” is not intended to limit thepresent disclosure to polynucleotides comprising DNA. Those of ordinaryskill in the art will recognize that polynucleotides, can compriseribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides disclosed herein also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, hairpins, stem-and-loop structures, and the like.

III. Expression Cassettes

The male fertility polynucleotides disclosed herein can be provided inexpression cassettes for expression in an organism of interest. Thecassette can include 5′ and 3′ regulatory sequences operably linked to amale fertility polynucleotide as disclosed herein. “Operably linked” isintended to mean a functional linkage between two or more elements. Forexample, an operable linkage between a polynucleotide of interest and aregulatory sequence (e.g., a promoter) is a functional link that allowsfor expression of the polynucleotide of interest. Operably linkedelements may be contiguous or non-contiguous. When used to refer to thejoining of two protein coding regions, by operably linked is intendedthat the coding regions are in the same reading frame.

The expression cassettes disclosed herein may include in the 5′-3′direction of transcription, a transcriptional and translationalinitiation region (i.e., a promoter), a polynucleotide of interest, anda transcriptional and translational termination region (i.e.,termination region) functional in the host cell (i.e., the plant).Expression cassettes are also provided with a plurality of restrictionsites and/or recombination sites for insertion of the male fertilitypolynucleotide to be under the transcriptional regulation of theregulatory regions described elsewhere herein. The regulatory regions(i.e., promoters, transcriptional regulatory regions, and translationaltermination regions) and/or the polynucleotide of interest may benative/analogous to the host cell or to each other. Alternatively, theregulatory regions and/or the polynucleotide of interest may beheterologous to the host cell or to each other. As used herein,“heterologous” in reference to a polynucleotide or polypeptide sequenceis a sequence that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous polynucleotide isfrom a species different from the species from which the polynucleotidewas derived, or, if from the same/analogous species, one or both aresubstantially modified from their original form and/or genomic locus, orthe promoter is not the native promoter for the operably linkedpolynucleotide. As used herein, a chimeric polynucleotide comprises acoding sequence operably linked to a transcription initiation regionthat is heterologous to the coding sequence.

In certain embodiments the polynucleotides disclosed herein can bestacked with any combination of polynucleotide sequences of interest orexpression cassettes as disclosed elsewhere herein. For example, themale fertility polynucleotides disclosed herein may be stacked with anyother polynucleotides encoding male-gamete disruptive polynucleotides orpolypeptides, cytotoxins, markers, or other male fertility sequences asdisclosed elsewhere herein. The stacked polynucleotides may be operablylinked to the same promoter as the male fertility polynucleotide, or maybe operably linked to a separate promoter polynucleotide.

As described elsewhere herein, expression cassettes may comprise apromoter operably linked to a polynucleotide of interest, along with acorresponding termination region. The termination region may be nativeto the transcriptional initiation region, may be native to the operablylinked male fertility polynucleotide of interest or with the malefertility promoter sequences, may be native to the plant host, or may bederived from another source (i.e., foreign or heterologous). Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

Where appropriate, the polynucleotides of interest may be optimized forincreased expression in the transformed plant. That is, thepolynucleotides can be synthesized using plant-preferred codons forimproved expression. See, for example, Campbell and Gowri (1990) PlantPhysiol. 92:1-11 for a discussion of host-preferred codon usage. Methodsare available in the art for synthesizing plant-preferred genes. See,for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al.(1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallieet al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf MosaicVirus) (Johnson et al. (1986) Virology 154:9-20), and humanimmunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991)Nature 353:90-94); untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) inMolecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); andmaize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991)Virology 81:382-385). See also, Della-Cioppa et al. (1987) PlantPhysiol. 84:965-968. Other methods known to enhance translation can alsobe utilized, for example, introns, and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A. Expression Cassettes Comprising a Male Fertility Polynucleotide Inparticular embodiments, the expression cassettes disclosed hereincomprise a promoter operably linked to a male fertility polynucleotide,or active fragment or variant thereof, as disclosed herein. In certainembodiments, a male fertility promoter or an active fragment or variantthereof, is operably linked to a male fertility polynucleotide disclosedherein, such as the male fertility polynucleotide set forth in SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, or 17, or an active fragment or variantthereof.

In certain embodiments, plant promoters can preferentially initiatetranscription in certain tissues, such as stamen, anther, filament, andpollen, or developmental growth stages, such as sporogenous tissue,microspores, and microgametophyte. Such plant promoters are referred toas “tissue-preferred”, “cell type-preferred”, or “growth-stagepreferred”. Promoters which initiate transcription only in certaintissue are referred to as “tissue-specific”. Likewise, promoters whichinitiate transcription only at certain growth stages are referred to as“growth stage-specific”. A “cell type-specific” promoter drivesexpression only in certain cell types in one or more organs, forexample, stamen cells, or individual cell types within the stamen suchas anther, filament, or pollen cells.

Male fertility polynucleotides disclosed herein, and active fragmentsand variants thereof, can be operably linked to male-tissue-specific ormale-tissue-preferred promoters including, for example, stamen-specificor stamen-preferred promoters, anther-specific or anther-preferredpromoters, pollen-specific or pollen-preferred promoters,tapetum-specific promoters or tapetum-preferred promoters, and the like.Promoters can be selected based on the desired outcome. For example, thepolynucleotides of interest can be operably linked to constitutive,tissue-preferred, growth stage-preferred, or other promoters forexpression in plants.

In one embodiment, the promoters may be those which preferentiallyexpress a polynucleotide of interest in the male tissues of the plant.No particular male fertility tissue-preferred promoter must be used inthe process, and any of the many such promoters known to one skilled inthe art may be employed. One such promoter is the 5126 promoter, whichpreferentially directs expression of the polynucleotide to which it islinked to male tissue of the plants, as described in U.S. Pat. Nos.5,837,851 and 5,689,051. Other examples include the maize Ms45 promoterdescribed at U.S. Pat. No. 6,037,523; SF3 promoter described at U.S.Pat. No. 6,452,069; the BS92-7 promoter described at WO 02/063021; aSGB6 regulatory element described at U.S. Pat. No. 5,470,359; the TA29promoter (Koltunow, et al., (1990) Plant Cell 2:1201-1224; Goldberg, etal., (1993) Plant Cell 5:1217-1229 and U.S. Pat. No. 6,399,856); thetype 2 metallothionein-like gene promoter (Charbonnel-Campaa, et al.,Gene (2000) 254:199-208) and the Brassica Bca9 promoter (Lee, et al.,(2003) Plant Cell Rep. 22:268-273).

In some embodiments, expression cassettes comprise male-gamete-preferredpromoters operably linked to a male fertility polynucleotide.Male-gamete-preferred promoters include the PG47 promoter (U.S. Pat.Nos. 5,412,085; 5,545,546; Plant J 3(2):261-271 (1993)), as well as ZM13promoter (Hamilton, et al., (1998) Plant Mol. Biol. 38:663-669); actindepolymerizing factor promoters (such as Zmabp1, Zmabp2; see, forexample Lopez, et al., (1996). Proc. Natl. Acad. Sci. USA 93:7415-7420);the promoter of the maize pectin methylesterase-like gene, ZmC5(Wakeley, et al., (1998) Plant Mol. Biol. 37:187-192); the profilin genepromoter Zmpro1 (Kovar, et al., (2000) The Plant Cell 12:583-598); thesulphated pentapeptide phytosulphokine gene ZmPSK1 (Lorbiecke, et al.,(2005) Journal of Experimental Botany 56(417):1805-1819); the promoterof the calmodulin binding protein Mpcbp (Reddy, et al., (2000) J. Biol.Chem. 275(45):35457-70).

As disclosed herein, constitutive promoters include, for example, thecore promoter of the Rsyn7 promoter and other constitutive promotersdisclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35Spromoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroyet al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al.(1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) PlantMol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet.81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALSpromoter (U.S. Pat. No. 5,659,026), and the like. Other constitutivepromoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144;5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and6,177,611.

“Seed-preferred” promoters include both those promoters active duringseed development such as promoters of seed storage proteins as well asthose promoters active during seed germination. See Thompson et al.(1989) BioEssays 10:108, herein incorporated by reference. Suchseed-preferred promoters include, but are not limited to, Cim1(cytokinin-induced message); cZ19B1 (maize 19 kDa zein); mi1ps(myo-inositol-1-phosphate synthase) (see WO 00/11177 and U.S. Pat. No.6,225,529; herein incorporated by reference). Gamma-zein is anendosperm-specific promoter. Globulin-1 (Glob-1) is a representativeembryo-specific promoter. For dicots, seed-specific promoters include,but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybeanlectin, cruciferin, and the like. For monocots, seed-specific promotersinclude, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDazein, gamma-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. Seealso WO 00/12733, where seed-preferred promoters from end1 and end2genes are disclosed; herein incorporated by reference. Additional embryospecific promoters are disclosed in Sato et al. (1996) Proc. Natl. Acad.Sci. 93:8117-8122; Nakase et al. (1997) Plant J 12:235-46; andPostma-Haarsma et al. (1999) Plant Mol. Biol. 39:257-71. Additionalendosperm specific promoters are disclosed in Albani et al. (1984) EMBO3:1405-15; Albani et al. (1999) Theor. Appl. Gen. 98:1253-62; Albani etal. (1993) Plant J. 4:343-55; Mena et al. (1998) The Plant Journal116:53-62, and Wu et al. (1998) Plant Cell Physiology 39:885-889.

Dividing cell or meristematic tissue-preferred promoters have beendisclosed in Ito et al. (1994) Plant Mol. Biol. 24:863-878; Reyad et al.(1995) Mo. Gen. Genet. 248:703-711; Shaul et al. (1996) Proc. Natl.Acad. Sci. 93:4868-4872; Ito et al. (1997) Plant J. 11:983-992; andTrehin et al. (1997) Plant Mol. Biol. 35:667-672.

Stress inducible promoters include salt/water stress-inducible promoterssuch as P5CS (Zang et al. (1997) Plant Sciences 129:81-89);cold-inducible promoters, such as, cor15a (Hajela et al. (1990) PlantPhysiol. 93:1246-1252), cor15b (Wlihelm et al. (1993) Plant Mol Biol23:1073-1077), wsc120 (Ouellet et al. (1998) FEBS Lett. 423-324-328),ci7 (Kirch et al. (1997) Plant Mol Biol. 33:897-909), ci21A (Schneideret al. (1997) Plant Physiol. 113:335-45); drought-inducible promoters,such as, Trg-31 (Chaudhary et al (1996) Plant Mol. Biol. 30:1247-57),rd29 (Kasuga et al. (1999) Nature Biotechnology 18:287-291); osmoticinducible promoters, such as, Rab 17 (Vilardell et al. (1991) Plant Mol.Biol. 17:985-93) and osmotin (Raghothama et al. (1993) Plant Mol Biol23:1117-28); and, heat inducible promoters, such as, heat shock proteins(Barros et al. (1992) Plant Mol. 19:665-75; Marrs et al. (1993) Dev.Genet. 14:27-41), and smHSP (Waters et al. (1996) J. Experimental Botany47:325-338). Other stress-inducible promoters include rip2 (U.S. Pat.No. 5,332,808 and U.S. Publication No. 2003/0217393) and rp29a(Yamaguchi-Shinozaki et al. (1993) Mol. Gen. Genetics 236:331-340).

As discussed elsewhere herein, the expression cassettes comprising malefertility polynucleotides may be stacked with other polynucleotides ofinterest. Any polynucleotide of interest may be stacked with the malefertility polynucleotide, including for example, male-gamete-disruptivepolynucleotides and marker polynucleotides.

Male fertility polynucleotides disclosed herein may be stacked in orwith expression cassettes comprising a promoter operably linked to apolynucleotide which is male-gamete-disruptive; that is, apolynucleotide which interferes with the function, formation, ordispersal of male gametes. A male-gamete-disruptive polynucleotide canoperate to prevent function, formation, or dispersal of male gametes byany of a variety of methods. By way of example but not limitation, thiscan include use of polynucleotides which encode a gene product such asDAM-methylase or barnase (See, for example, U.S. Pat. No. 5,792,853 or5,689,049; PCT/EP89/00495); encode a gene product which interferes withthe accumulation of starch or affects osmotic balance in pollen (See,for example, U.S. Pat. Nos. 7,875,764; 8,013,218; 7,696,405); inhibitformation of a gene product important to male gamete function,formation, or dispersal (See, for example, U.S. Pat. Nos. 5,859,341;6,297,426); encode a gene product which combines with another geneproduct to prevent male gamete formation or function (See U.S. Pat. Nos.6,162,964; 6,013,859; 6,281,348; 6,399,856; 6,248,935; 6,750,868;5,792,853); are antisense to, or cause co-suppression of, a genecritical to male gamete function, formation, or dispersal (See U.S. Pat.Nos. 6,184,439; 5,728,926; 6,191,343; 5,728,558; 5,741,684); interferewith expression of a male fertility polynucleotide through use ofhairpin formations (Smith et al. (2000) Nature 407:319-320; WO 99/53050and WO 98/53083) or the like.

Male-gamete-disruptive polynucleotides include dominant negative genessuch as methylase genes and growth-inhibiting genes. See, U.S. Pat. No.6,399,856. Dominant negative genes include diphtheria toxin A-chain gene(Czako and An (1991) Plant Physiol. 95 687-692; Greenfield et al. (1983)PNAS 80:6853); cell cycle division mutants such as CDC in maize(Colasanti et al. (1991) PNAS 88: 3377-3381); the WT gene (Farmer et al.(1994) Mol. Genet. 3:723-728); and P68 (Chen et al. (1991) PNAS88:315-319).

Further examples of male-gamete-disruptive polynucleotides include, butare not limited to, pectate lyase gene pelE from Erwinia chrysanthermi(Kenn et al (1986) J. Bacteriol. 168:595); CytA toxin gene from Bacillusthuringiensis Israeliensis (McLean et al (1987) J. Bacteriol. 169:1017(1987), U.S. Pat. No. 4,918,006); DNAses, RNAses, proteases, orpolynucleotides expressing anti-sense RNA. A male-gamete-disruptivepolynucleotide may encode a protein involved in inhibiting pollen-stigmainteractions, pollen tube growth, fertilization, or a combinationthereof.

Male fertility polynucleotides disclosed herein may be stacked withexpression cassettes disclosed herein comprising a promoter operablylinked to a polynucleotide of interest encoding a reporter or markerproduct. Examples of suitable reporter polynucleotides known in the artcan be found in, for example, Jefferson et al. (1991) in Plant MolecularBiology Manual, ed. Gelvin et al. (Kluwer Academic Publishers), pp.1-33; DeWet et al. Mol. Cell. Biol. 7:725-737 (1987); Goff et al. EMBOJ. 9:2517-2522 (1990); Kain et al. BioTechniques 19:650-655 (1995); andChiu et al. Current Biology 6:325-330 (1996). In certain embodiments,the polynucleotide of interest encodes a selectable reporter. These caninclude polynucleotides that confer antibiotic resistance or resistanceto herbicides. Examples of suitable selectable marker polynucleotidesinclude, but are not limited to, genes encoding resistance tochloramphenicol, methotrexate, hygromycin, streptomycin, spectinomycin,bleomycin, sulfonamide, bromoxynil, glyphosate, and phosphinothricin.

In some embodiments, the expression cassettes disclosed herein comprisea polynucleotide of interest encoding scorable or screenable markers,where presence of the polynucleotide produces a measurable product.Examples include a β-glucuronidase, or uidA gene (GUS), which encodes anenzyme for which various chromogenic substrates are known (for example,U.S. Pat. Nos. 5,268,463 and 5,599,670); chloramphenicol acetyltransferase, and alkaline phosphatase. Other screenable markers includethe anthocyanin/flavonoid polynucleotides including, for example, aR-locus polynucleotide, which encodes a product that regulates theproduction of anthocyanin pigments (red color) in plant tissues, thegenes which control biosynthesis of flavonoid pigments, such as themaize C1 and C2, the B gene, the p1 gene, and the bronze locus genes,among others. Further examples of suitable markers encoded bypolynucleotides of interest include the cyan fluorescent protein (CYP)gene, the yellow fluorescent protein gene, a lux gene, which encodes aluciferase, the presence of which may be detected using, for example,X-ray film, scintillation counting, fluorescent spectrophotometry,low-light video cameras, photon counting cameras or multiwellluminometry, a green fluorescent protein (GFP), and DsRed2 where plantcells transformed with the marker gene are red in color, and thusvisually selectable. Additional examples include a p-lactamase geneencoding an enzyme for which various chromogenic substrates are known(e.g., PADAC, a chromogenic cephalosporin), a xylE gene encoding acatechol dioxygenase that can convert chromogenic catechols, anα-amylase gene, and a tyrosinase gene encoding an enzyme capable ofoxidizing tyrosine to DOPA and dopaquinone, which in turn condenses toform the easily detectable compound melanin.

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su et al. (2004)Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. CellScience 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), andyellow florescent protein (PhiYFP™ from Evrogen, see, Bolte et al.(2004) J. Cell Science 117:943-54). For additional selectable markers,see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511;Christopherson et al. (1992) Proc. Natl. Acad Sci. USA 89:6314-6318; Yaoet al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol.6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.(1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge etal. (1988) Cell 52:713-722; Deuschle et at (1989) Proc. Natl. Acad. Sci.USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl.Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidtet at (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et at (1992) Proc. Natl. Acad. Sci. USA89:5547-5551; Oliva et at (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724. Such disclosures are herein incorporated by reference. Theabove list of selectable marker genes is not meant to be limiting. Anyselectable marker gene can be used in the compositions and methodsdisclosed herein.

In some embodiments, the expression cassettes disclosed herein comprisea first polynucleotide of interest encoding a male fertilitypolynucleotide operably linked to a first promoter polynucleotidestacked with a second polynucleotide of interest encoding amale-gamete-disruptive gene product operably linked to a maletissue-preferred promoter polynucleotide. In other embodiments, theexpression cassettes described herein may also be stacked with a thirdpolynucleotide of interest encoding a marker polynucleotide operablylinked to a third promoter polynucleotide.

In specific embodiments, the expression cassettes disclosed hereincomprise a first polynucleotide of interest encoding a wheat malefertility gene disclosed herein, such as Ms22, Ms26, or Ms45 operablylinked to a constitutive promoter, such as the cauliflower mosaic virus(CaMV) 35S promoter. The expression cassettes may further comprise asecond polynucleotide of interest encoding a male-gamete-disruptive geneproduct operably linked to a male tissue-preferred promoter. In certainembodiments, the expression cassettes disclosed herein may furthercomprise a third polynucleotide of interest encoding a marker gene, suchas the phosphinothricin acetyltransferase (PAT) gene from Streptomycesviridochomagenes operably linked to a constitutive promoter, such as thecauliflower mosaic virus (CaMV) 35S promoter.

IV. Plants

A. Plants Having Altered Levels/Activity of Male Fertility Polypeptide

Further provided are plants having altered levels and/or activities of amale fertility polypeptide and/or altered levels of male fertility. Insome embodiments, the plants disclosed herein have stably incorporatedinto their genomes a heterologous male fertility polynucleotide, oractive fragments or variants thereof, as disclosed herein. Thus, plants,plant cells, plant parts, and seeds are provided which comprise at leastone heterologous male fertility polynucleotide as set forth in any oneof SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17 or any active fragmentsor variants disclosed herein.

Plants are further provided comprising the expression cassettesdisclosed herein comprising a male fertility polynucleotide operablylinked to a promoter that is active in the plant. In some embodiments,expression of the male fertility polynucleotide modulates male fertilityof the plant. In certain embodiments, expression of the male fertilitypolynucleotide increases male fertility of the plant. For example,plants are provided comprising an expression cassette comprising an Ms22polynucleotide as set forth in SEQ ID NO: 1, 3, or 5, or an activefragment or variant thereof, operably linked to a constitutive promoter,such as the CaMV 35S promoter. Upon expression of the Ms22polynucleotide, male fertility of the plant is increased.

In certain embodiments, expression cassettes comprising a heterologousmale fertility polynucleotide as disclosed herein, or an active fragmentor variant thereof, operably linked to a promoter active in a plant, areprovided to a male sterile plant. Upon expression of the heterologousmale fertility polynucleotide, the male fertility of the plant isrestored. In specific embodiments, the plants disclosed herein comprisean expression cassette comprising a heterologous male fertilitypolynucleotide as disclosed herein, or an active fragment or variantthereof, operably linked to a promoter, stacked with one or moreexpression cassettes comprising a polynucleotide of interest operablylinked to a promoter active in the plant. For example, the stackedpolynucleotide of interest can comprise a male-gamete-disruptivepolynucleotide and/or a marker polynucleotide.

Plants disclosed herein may also comprise stacked expression cassettesdescribed herein comprising at least two polynucleotides such that theat least two polynucleotides are inherited together in more than 50% ofmeioses, i.e., not randomly. Accordingly, when a plant or plant cellcomprising stacked expression cassettes with two polynucleotidesundergoes meiosis, the two polynucleotides segregate into the sameprogeny (daughter) cell. In this manner, stacked polynucleotides willlikely be expressed together in any cell for which they are present. Forexample, a plant may comprise an expression cassette comprising a malefertility polynucleotide stacked with an expression cassette comprisinga male-gamete-disruptive polynucleotide such that the male fertilitypolynucleotide and the male-gamete-disruptive polynucleotide areinherited together. Specifically, a male sterile plant could comprise anexpression cassette comprising a male fertility polynucleotide disclosedherein operably linked to a constitutive promoter, stacked with anexpression cassette comprising a male-gamete-disruptive polynucleotideoperably linked to a male tissue-preferred promoter, such that the plantproduces mature pollen grains. However, in such a plant, development ofthe daughter pollen cells comprising the male fertility polynucleotidewill be prevented by expression of the male-gamete-disruptivepolynucleotide.

B. Plants and Methods of Introduction

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which a plant can be regenerated, plantcalli, plant clumps, and plant cells that are intact in plants or partsof plants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, grain and the like. As used herein “grain” is intended themature seed produced by commercial growers for purposes other thangrowing or reproducing the species. Progeny, variants, and mutants ofthe regenerated plants are also included within the scope of thedisclosure, provided that these parts comprise the introduced nucleicacid sequences.

The methods disclosed herein comprise introducing a polypeptide orpolynucleotide into a plant cell. “Introducing” is intended to meanpresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell. Themethods disclosed herein do not depend on a particular method forintroducing a sequence into the host cell, only that the polynucleotideor polypeptides gains access to the interior of at least one cell of thehost. Methods for introducing polynucleotide or polypeptides into hostcells (i.e., plants) are known in the art and include, but are notlimited to, stable transformation methods, transient transformationmethods, and virus-mediated methods.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a host (i.e., a plant) integrates into thegenome of the plant and is capable of being inherited by the progenythereof. “Transient transformation” is intended to mean that apolynucleotide is introduced into the host (i.e., a plant) and expressedtemporally or a polypeptide is introduced into a host (i.e., a plant).

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing polypeptides andpolynucleotides into plant cells include microinjection (Crossway et al.(1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986)Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediatedtransformation (Townsend et al., U.S. Pat. No. 5,563,055; Zhao et al.,U.S. Pat. No. 5,981,840), direct gene transfer (Paszkowski et al. (1984)EMBO J. 3:2717-2722), and ballistic particle acceleration (see, forexample, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et at, U.S. Pat.No. 5,879,918; Tomes et U.S. Pat. No. 5,886,244; Bidney et al., U.S.Pat. No. 5,932,782; Tomes et al. (1995) “Direct DNA Transfer into IntactPlant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, andOrgan Culture: Fundamental Methods, ed. Gamborg and Phillips(Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology6:923-926); and Lecl transformation (WO 00/28058). Also see Weissingeret al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987)Particulate Science and Technology 5:27-37 (onion); Christou et al.(1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl.Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes,U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and5,324,646; Tomes et al. (1995) “Direct DNA Transfer into Intact PlantCells via Microprojectile Bombardment,” in Plant Cell, Tissue, and OrganCulture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin)(maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm etal. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren etal. (1984) Nature (London) 311:763-764; Bowen et al., U.S. Pat. No.5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

In specific embodiments, the male fertility polynucleotides orexpression cassettes disclosed herein can be provided to a plant using avariety of transient transformation methods. Such transienttransformation methods include, but are not limited to, the introductionof the male fertility polypeptide or variants and fragments thereofdirectly into the plant or the introduction of a male fertilitytranscript into the plant. Such methods include, for example,microinjection or particle bombardment. See, for example, Crossway etal. (1986) Mol Gen. Genet. 202:179-185; Nomura et al. (1986) Plant Sci.44:53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 andHush et al. (1994) The Journal of Cell Science 107:775-784, all of whichare herein incorporated by reference. Alternatively, the male fertilitypolynucleotide or expression cassettes disclosed herein can betransiently transformed into the plant using techniques known in theart. Such techniques include viral vector system and the precipitationof the polynucleotide in a manner that precludes subsequent release ofthe DNA. Thus, the transcription from the particle-bound DNA can occur,but the frequency with which it is released to become integrated intothe genome is greatly reduced. Such methods include the use of particlescoated with polyethylimine (PEI; Sigma #P3143).

In other embodiments, the male fertility polynucleotides or expressioncassettes disclosed herein may be introduced into plants by contactingplants with a virus or viral nucleic acids. Generally, such methodsinvolve incorporating a nucleotide construct of disclosed herein withina viral DNA or RNA molecule. It is recognized that a male fertilitysequence disclosed herein may be initially synthesized as part of aviral polyprotein, which later may be processed by proteolysis in vivoor in vitro to produce the desired recombinant protein. Methods forintroducing polynucleotides into plants and expressing a protein encodedtherein, involving viral DNA or RNA molecules, are known in the art.See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785,5,589,367, 5,316,931, and Porta et al. (1996) Molecular Biotechnology5:209-221; herein incorporated by reference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference. Briefly,the polynucleotide disclosed herein can be contained in transfercassette flanked by two non-identical recombination sites. The transfercassette is introduced into a plant having stably incorporated into itsgenome a target site which is flanked by two non-identical recombinationsites that correspond to the sites of the transfer cassette. Anappropriate recombinase is provided and the transfer cassette isintegrated at the target site. The polynucleotide of interest is therebyintegrated at a specific chromosomal position in the plant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andpollinated with either the same transformed strain or different strains,and the resulting progeny having desired expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present disclosure provides transformed seed (alsoreferred to as “transgenic seed”) having a male fertility polynucleotidedisclosed herein, for example, an expression cassette disclosed herein,stably incorporated into their genome. Seed comprising any expressioncassette disclosed herein can be sorted based on size parameters,including but not limited to, seed length, seed width, seed density, orany combination thereof.

The male fertility polynucleotides and expression cassettes disclosedherein may be used for transformation of any plant species, including,but not limited to, monocots and dicots. Examples of plant species ofinterest include, but are not limited to, corn (Zea mays), Brassica sp.(e.g., B. napus, B. rapa, B. juncea), alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum luberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables, ornamentals, grasses and conifers.

In particular embodiments, wheat plants are used in the methods andcompositions disclosed herein. As used herein, the term “wheat” refersto any species of the genus Triticum, including progenitors thereof, aswell as progeny thereof produced by crosses with other species. Wheatincludes “hexaploid wheat” which has genome organization of AABBDD,comprised of 42 chromosomes, and “tetraploid wheat” which has genomeorganization of AABB, comprised of 28 chromosomes. Hexaploid wheatincludes T. aestivum, T. spelta, T. mocha, T. compactum, Tsphaerococcum, T. vavilovii, and interspecies cross thereof. Tetraploidwheat includes T. durum (also referred to as durum wheat or Triticumturgid=ssp. durum), T. dicoccoides, T. dicoccum, T. polonicum, andinterspecies cross thereof. In addition, the term “wheat” includespossible progenitors of hexaploid or tetraploid Triticum sp. such as T.uartu, T. monococcum or T. boeoticum for the A genome, Aegilopsspeltoides for the B genome, and T. tauschii (also known as Aegilopssquarrosa or Aegilops tauschii) for the D genome. A wheat cultivar foruse in the present disclosure may belong to, but is not limited to, anyof the above-listed species. Also encompassed are plants that areproduced by conventional techniques using Triticum sp. as a parent in asexual cross with a non-Triticum species, such as rye Secale cereale,including but not limited to Triticale. In some embodiments, the wheatplant is suitable for commercial production of grain, such as commercialvarieties of hexaploid wheat or durum wheat, having suitable agronomiccharacteristics which are known to those skilled in the art.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the present methods andcompositions include, for example, pines such as loblolly pine (Pinustaeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa),lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata);Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis);Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firssuch as silver fir (Abies amabilis) and balsam fir (Abies balsamea); andcedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). In specific embodiments, plants disclosedherein are crop plants (for example, corn, alfalfa, sunflower, Brassica,soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco,etc.). In other embodiments, corn and soybean plants are optimal, and inyet other embodiments corn plants are optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

Typically, an intermediate host cell will be used in the practice of themethods and compositions disclosed herein to increase the copy number ofthe cloning vector. With an increased copy number, the vector containingthe nucleic acid of interest can be isolated in significant quantitiesfor introduction into the desired plant cells. In one embodiment, plantpromoters that do not cause expression of the polypeptide in bacteriaare employed.

Prokaryotes most frequently are represented by various strains of E.coli; however, other microbial strains may also be used. Commonly usedprokaryotic control sequences which are defined herein to includepromoters for transcription initiation, optionally with an operator,along with ribosome binding sequences, include such commonly usedpromoters as the beta lactamase (penicillinase) and lactose (lac)promoter systems (Chang et al. (1977) Nature 198:1056), the tryptophan(trp) promoter system (Goeddel et al. (1980) Nucleic Acids Res. 8:4057)and the lambda derived P L promoter and N-gene ribosome binding site(Shimatake et al. (1981) Nature 292:128). The inclusion of selectionmarkers in DNA vectors transfected in E. coli. is also useful. Examplesof such markers include genes specifying resistance to ampicillin,tetracycline, or chloramphenicol.

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing a protein disclosed herein areavailable using Bacillus sp. and Salmonella (Palva et al. (1983) Gene22:229-235); Mosbach et al. (1983) Nature 302:543-545).

In some embodiments, the expression cassette or male fertilitypolynucleotides disclosed herein are maintained in a hemizygous state ina plant. Hemizygosity is a genetic condition existing when there is onlyone copy of a gene (or set of genes) with no allelic counterpart on thesister chromosome. In certain embodiments, the expression cassettesdisclosed herein comprise a first promoter operably linked to a malefertility polynucleotide which is stacked with a male-gamete-disruptivepolynucleotide operably linked to a male tissue-preferred promoter, andsuch expression cassettes are introduced into a male sterile plant in ahemizygous condition. When the male fertility polynucleotide isexpressed, the plant is able to successfully produce mature pollengrains because the male fertility polynucleotide restores the plant to afertile condition. Given the hemizygous condition of the expressioncassette, only certain daughter cells will inherit the expressioncassette in the process of pollen grain formation. The daughter cellsthat inherit the expression cassette containing the male fertilitypolynucleotide will not develop into mature pollen grains due to themale tissue-preferred expression of the stacked encodedmale-gamete-disruptive gene product. Those pollen grains that do notinherit the expression cassette will continue to develop into maturepollen grains and be functional, but will not contain the male fertilitypolynucleotide of the expression cassette and therefore will nottransmit the male fertility polynucleotide to progeny through pollen.

V. Modulating the Concentration and/or Activity of Male FertilityPolypeptides

A method for modulating the concentration and/or activity of the malefertility polypeptides disclosed herein in a plant is provided. The term“influences” or “modulates”, as used herein with reference to theconcentration and/or activity of the male fertility polypeptides, refersto any increase or decrease in the concentration and/or activity of themale fertility polypeptides when compared to an appropriate control. Ingeneral, concentration and/or activity of a male fertility polypeptidedisclosed herein is increased or decreased by at least 1%, 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to a native control plant,plant part, or cell. Modulation as disclosed herein may occur duringand/or subsequent to growth of the plant to the desired stage ofdevelopment. In specific embodiments, the male fertility polypeptidesdisclosed herein are modulated in monocots, particularly wheat.

A variety of methods can be employed to assay for modulation in theconcentration and/or activity of a male fertility polypeptide. Forinstance, the expression level of the male fertility polypeptide may bemeasured directly, for example, by assaying for the level of the malefertility polypeptide in the plant (i.e., Western or Northern blot), orindirectly, for example, by assaying the male fertility activity of themale fertility polypeptide in the plant. Methods for measuring the malefertility activity are described elsewhere herein. In specificembodiments, modulation of male fertility polypeptide concentrationand/or activity comprises the modulation (i.e., an increase or adecrease) in the level of male fertility polypeptide in the plant.Methods to measure the level and/or activity of male fertilitypolypeptides are known in the art and are discussed elsewhere herein. Instill other embodiments, the level and/or activity of the male fertilitypolypeptide is modulated in vegetative tissue, in reproductive tissue,or in both vegetative and reproductive tissue.

In one embodiment, the activity and/or concentration of the malefertility polypeptide is increased by introducing the polypeptide or thecorresponding male fertility polynucleotide into the plant.Subsequently, a plant having the introduced male fertility sequence isselected using methods known to those of skill in the art such as, butnot limited to, Southern blot analysis, DNA sequencing, PCR analysis, orphenotypic analysis. In certain embodiments, marker polynucleotides areintroduced with the male fertility polynucleotide to aid in selection ofa plant having or lacking the male fertility polynucleotide disclosedherein. A plant or plant part altered or modified by the foregoingembodiments is grown under plant forming conditions for a timesufficient to modulate the concentration and/or activity of the malefertility polypeptide in the plant. Plant forming conditions are wellknown in the art.

As discussed elsewhere herein, many methods are known the art forproviding a polypeptide to a plant including, but not limited to, directintroduction of the polypeptide into the plant, or introducing into theplant (transiently or stably) a polynucleotide construct encoding a malefertility polypeptide. It is also recognized that the methods disclosedherein may employ a polynucleotide that is not capable of directing, inthe transformed plant, the expression of a protein or an RNA. Thus, thelevel and/or activity of a male fertility polypeptide may be increasedby altering the gene encoding the male fertility polypeptide or itspromoter. See, e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling et al.,PCT/US93/03868. Therefore mutagenized plants that carry mutations inmale fertility genes, where the mutations increase expression of themale fertility gene or increase the activity of the encoded malefertility polypeptide are provided.

In other embodiments, the concentration and/or activity of a malefertility polypeptide is increased by introduction into a plant of anexpression cassette comprising a male fertility polynucleotide (e.g. SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17), or an active fragment orvariant thereof, as disclosed elsewhere herein. The male fertilitypolynucleotide may be operably linked to promoter that is heterologousto the plant or native to the plant. By increasing the concentrationand/or activity of a male fertility polypeptide in a plant, the malefertility of the plant is likewise increased. Thus, the male fertilityof a plant can be increased by increasing the concentration and/oractivity of a male fertility polypeptide. For example, male fertilitycan be restored to a male sterile plant by increasing the concentrationand/or activity of a male fertility polypeptide.

It is also recognized that the level and/or activity of the polypeptidemay be modulated by employing a polynucleotide that is not capable ofdirecting, in a transformed plant, the expression of a protein or anRNA. For example, the polynucleotides disclosed herein may be used todesign polynucleotide constructs that can be employed in methods foraltering or mutating a genomic nucleotide sequence in an organism. Suchpolynucleotide constructs include, but are not limited to, RNA:DNAvectors, RNA:DNA mutational vectors, RNA:DNA repair vectors,mixed-duplex oligonucleotides, self-complementary RNA:DNAoligonucleotides, and recombinogenic oligonucleobases. Such nucleotideconstructs and methods of use are known in the art. See, U.S. Pat. Nos.5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972; and 5,871,984;all of which are herein incorporated by reference. See also, WO98/49350, WO 99/07865, WO 99/25821, and Beetham et al. (1999) Proc.Natl. Acad. Sci. USA 96:8774-8778; herein incorporated by reference. Itis therefore recognized that methods disclosed herein do not depend onthe incorporation of the entire polynucleotide into the genome, onlythat the plant or cell thereof is altered as a result of theintroduction of the polynucleotide into a cell.

In one embodiment, the genome may be altered following the introductionof the polynucleotide into a cell. For example, the polynucleotide, orany part thereof, may incorporate into the genome of the plant.Alterations to the genome disclosed herein include, but are not limitedto, additions, deletions, and substitutions of nucleotides into thegenome. While the methods disclosed herein do not depend on additions,deletions, and substitutions of any particular number of nucleotides, itis recognized that such additions, deletions, or substitutions comprisesat least one nucleotide.

TABLE 1 Summary of SEQ ID NOS SEQ ID: Description 1 Wheat (T. urartu, Agenome) Ms22 polynucleotide 2 Wheat (T. urartu, A genome) Ms22polypeptide 3 Wheat (Ae. Speltoides, B genome) Ms22 polynucleotide 4Wheat (Ae. Speltoides, B genome) Ms22 polypeptide 5 Wheat (Ae. Tauschii,D genome) Ms22 polynucleotide 6 Wheat (Ae. Tauschii, D genome) Ms22polypeptide 7 Wheat (T. urartu, A genome) Ms26 polynucleotide 8 Wheat(T. urartu, A genome) Ms26 polypeptide 9 Wheat (Ae. Speltoides, Bgenome) Ms26 polynucleotide 10 Wheat (Ae. Speltoides, B genome) Ms26polypeptide 11 Wheat (Ae. Tauschii, D genome) Ms26 polynucleotide 12Wheat (Ae. Tauschii, D genome) Ms26 polypeptide 13 Wheat (T. urartu, Agenome) Ms45 polynucleotide 14 Wheat (T. urartu, A genome) Ms45polypeptide 15 Wheat (Ae. Speltoides, B genome) Ms45 polynucleotide 16Wheat (Ae. Speltoides, B genome) Ms45 polypeptide 17 Wheat (Ae.Tauschii, D genome) Ms45 polynucleotide 18 Wheat (Ae. Tauschii, Dgenome) Ms45 polypeptide 19 Heme-binding domain of Ms26: FxxGxRxCxG 20Dioxygen binding domain A of Ms26 A/GGXD/ETT/S 21 MS26+ target site22-27 primers 28 Wheat A genome, FIG. 1 29 Wheat B genome, FIG. 1 30Wheat D genome, FIG. 1 31 Maize MS26, FIG. 1 32 Sorghum MS26, FIG. 1 33Rice MS26, FIG. 1 34-44 See FIG. 2 description 45-52 See FIG. 4description

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisdisclosure pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

EXPERIMENTAL Example 1. Identification of Male Fertility Polynucleotidesin Wheat

Male-fertility polynucleotides disclosed herein may be identified usingbioinformatic approaches. As an example, sequences putativelyrepresenting male-fertility genes in wheat are initially identified byan in silico search of proprietary databases using known fertility genesfrom other species, such as maize. Candidate ESTs are selected based onprotein-level homology to the reference sequences and consideration ofthe library from which the candidate sequence originated, e.g.representing expression in male reproductive tissue.

Based on the candidate EST sequences, primers are created and used toscreen a proprietary wheat BAC library. Super-pools identified arefurther screened with appropriate primers to identify specific BACclones comprising the ESTs.

Touchdown PCR may be performed (GeneAmp® PCR System 9700, AppliedBiosystems), using the following cycling parameters: 94° C. for 3 min(one cycle), 94° C. for 1 min, 55° C. for 1 min and 72° C. for 1 min 30s, (35 cycles), 72° C. for 7 min, and termination at 4° C. Pfu UltraHotstart™ DNA polymerase (Stratagene) may be preferred for its very lowaverage error rate (less than 0.5% per 500-bp fragment amplified).

Wheat insert DNA isolated from the BAC clones is digested for Southernblot confirmation using a candidate EST clone as a probe. BAC fragmentsare subcloned into pBluescript® (Stratagene Inc., La Jolla, Calif.).White colonies are grown in LB medium and transferred onto a membraneusing a dot-blot procedure. After denaturation the membrane is probedwith a candidate EST clone. Positive clones are identified andsequenced.

Example 2. Comparison of Wheat Male Fertility Polynucleotide with KnownSequences

TABLE 2 Global Identity of Wheat, Maize, and Rice Ms22 Polynucleotidesand Polypeptides T urartu Ae speltoides Ae tauschii Maize Rice MS22 Agenome B genome D genome ms22 ms22 T urartu 97.6* 99.0* 79.9* 81.0* Agenome Ae speltoides 98.5 98.1* 79.6* 81.0* B genome Ae tauschii 98.598.5 79.6* 81.5* D genome Maize ms22 89.2 89.2 89.2 83.6* Rice ms22 86.286.2 81.9 78.0 (Polynucleotide result is listed with an asterisk;polypeptide result is listed without an asterisk.)

TABLE 3 Global Identity of Wheat, Maize, and Rice Ms26 Polynucleotidesand Polypeptides T urartu Ae speltoides Ae tauschii Maize Rice MS26 Agenome B genome D genome ms26 ms26 T urartu 97.7* 97.9* 82.1* 81.8* Agenome Ae speltoides 99.1 97.9* 82.2* 82.3* B genome Ae tauschii 98.999.1 82.0* 82.8* D genome Maize ms26 89.0 89.0 88.8 80.5* Rice ms26 90.490.5 90.2 87.7 (Polynucleotide result is listed with an asterisk;polypeptide result is listed without an asterisk.)

TABLE 4 Global Identity of Wheat, Maize, and Rice Ms45 Polynucleotidesand Polypeptides T urartu Ae speltoides Ae tauschii Maize Rice MS45 Agenome B genome D genome ms45 ms45 T urartu 96.3* 98.5* 79.5* 78.0* Agenome Ae speltoides 99.3 96.7* 79.1* 78.6* B genome Ae tauschii 99.599.3 78.6* 78.9* D genome Maize ms45 81.6 81.8 81.1 76.9* Rice ms45 85.084.7 84.7 82.8 (Polynucleotide result is listed with an asterisk;polypeptide result is listed without an asterisk.)

Example 3. Wheat Transformation

Wheat transformation protocols are available to one of skill in the art.See, for example, He et al. (2010) J. Exp. Botany 61(6):1567-1581; Wu etal. (2008) Transgenic Res. 17:425-436; Nehra et al. (1994) Plant J.5(2):285-297; Rasco-Gaunt et al. (2001) J. Exp. Botany 52(357):865-874;Razzaq et al. (2011) African J. Biotech. 10(5):740-750.

Example 4. Directed Modification of MS26

This example describes methods to mutate wheat genes usingdouble-strand-break technologies to enable directed DNA modification orgene insertion via homologous recombination. More specifically, thisexample describes a method which includes, but is not limited to,delivery of a custom homing endonuclease, MS26+, to recognize, cleave,and mutate wheat chromosomal DNA through imprecise non-homologousend-joining (NHEJ) repair.

Vectors and Transformation:

Male fertility MS26 genes located within wheat genomes A, B and Dcontain a 22 base pair sequence (5′-GATGGTGACGTACGTGCCCTAC-3′; SEQ IDNO: 21) which is recognized by an MS26+ homing endonuclease as asubstrate for introducing a double strand break. The 22 bp MS26recognition site is present within the A, B and D wheat genomes andconserved across maize, sorghum and rice MS26 orthologous genes (FIG.1). The MS26+ homing endonuclease has been shown to generate mutationsin maize, rice and sorghum plants WO2013/066423, published on May 10,2013. To generate mutations in the genomic Ms26 genes in wheat plants,PHP42063 was introduced into wheat Fielder variety byAgrobacterium-mediated transformation methods similar to those described(Tamas-Nyitrai et al Plant Cell Cultures Protocols Methods in MolecularBiology 877, 2012, 357-384; He, et al., (2010) J. Exp. Botany61(6):1567-1581; Wu, et al., (2008) Transgenic Res. 17:425-436; Nehra,et al., (1994) Plant J. 5(2):285-297; Rasco-Gaunt, et al., (2001) J.Exp. Botany 52(357):865-874; Razzaq, et al., (2011) African J. Biotech.10(5):740-750).

PHP42063 contains a single chain MS26+ placed under the transcriptionalcontrol of the maize CAS1 promoter. The CAS1 promoter can betranscriptionally induced by either the sulfonylurea-safener, 2-CBSU, orby elevated temperature (U.S. patent application Ser. No. 13/896,437filed May 17, 2013). PHP42063 also contains a blue-fluorescence gene(CFP) regulated by the ZmEND2 promoter which is used as visual markerfor the selection of integration of the T-DNA into wheat cells. Inaddition, PHP42063 contains a copy of a red fluorescence gene regulatedby the maize Histone 2B promoter. A portion of the red fluorescence genein this construct was duplicated in a direct orientation, consisting oftwo fragments of the RFP gene with 369 bp of overlap. The two fragmentsare separated by a 136-bp spacer containing an MS26 target site. Bluefluorescing calli were selected and used for regeneration of wheatplants and grown in the greenhouse to maturity and seed set. Wheatplants containing TDNA insertions of PHP42063 were verified bycopy-number analysis. Four independent single or low-copy PHP42063transformed plants were selected for additional experimentation. Bluefluorescing immature embryos were harvested 14-20 days afterpollination, sterilized, placed on maintenance media and incubated inthe dark at 37 C for 24 hours. At the end of this period, embryosincubated at the elevated temperature were moved to room temperature(<26 C) and embryos were allowed to grow in the dark. Approximately 72hours after the initiation of treatment at elevated temperature, embryosincubated at 37 C begin to develop red fluorescing sectors. Thisobservation suggests that the heat inducible gene cassette, CAS1:MS26+,has resulted in double-strand-breaks at the MS26 target site between thetwo overlapping sequences of the RF-FP reporter, promotingintramolecular recombination and producing a functional RFP gene whichis revealed by the appearance of red fluorescing cells against abackground of blue fluorescence. Red fluorescing callus events wereselected for additional molecular characterization and plantregeneration.

Identification of Mutations at the TaMS26 Target Site in Plant Tissues.

Total genomic DNA was extracted from the heat treated and non-heattreated callus transformed with the MS26+ homing endonuclease and theregion surrounding the genomic target site was PCR amplified withPhusion® High Fidelity PCR Master Mix (New England Biolabs, M0531L)adding on the sequences necessary for amplicon-specific barcodes andIllumnia sequencing using “tailed” primers through two rounds of PCR.The primers used in the primary PCR reaction are shown in Table 5.

TABLE 5 PCR primer sequences Primer SEQ Target Site OrientationPrimary PCR Primer Sequence ID NO: MS26+ ForwardCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAA 22 Homing CCCGCGGAGGACGACGTGCTCEndonuclease MS26+ Reverse CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTCGT 23Homing CGGGGCCCCAGTTGTAC Endonuclease

The primers used in the secondary PCR reaction wereAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACG (forward, SEQ ID NO: 24) andCAAGCAGAAGACGGCATA (reverse, SEQ ID NO: 25). Genomic DNA extracted fromleaves of untransformed Fielder plants served as a negative control.

The resulting PCR amplifications were concentrated using a QiagenMinielute PCR purification spin column, electrophoresed on a 2% agarosegel and the appropriate amplifications were excised and purified with aQiagen Gel Extraction spin column. The concentration of the gel purifiedamplifications was measured with a Hoechst dye-based fluorometric assay,combined in an equimolar ratio, and single read 100 nucleotide-lengthdeep sequencing was performed on Illumina's Genome Analyzer IIx (ELIMBiopharmaceuticals, Inc.) with a 30-40% (v/v) spike of PhiX control v3(Illumina, FC-110-3001) to off-set sequence bias. Only those reads witha ≥1 nucleotide indel arising within a 6 nucleotide window centered overthe expected site of cleavage and not found in a similar level in thenegative control were classified as NHEJ mutations. The total numbers ofNHEJ mutations were then used to calculate the % mutant reads based onthe total number of reads of an appropriate length containing a perfectmatch to the barcode and forward primer.

The frequency of NHEJ mutations recovered by deep sequencing for thenon-heat treated and heat treated callus transformed with the MS26+homing endonuclease compared to the negative control is shown in Table6. The ten most prevalent types of NHEJ mutations recovered from theheat treated callus are shown in FIG. 2. These data suggest that theMS26+ homing endonuclease effectively introduced NHEJ mutations andalterations into the native MS26 wheat genes.

TABLE 6 Percent (%) mutant reads at the wheat MS26+ homing endonucleasetarget locus. Total Total Number of Number of % Mutant System MutantReads Reads Reads Untransformed Wheat 19 3,989,749 0.00% Genomic DNAControl Wheat Callus Transformed 304 4,069,593 0.007%  with MS26+ HomingEndonuclease (No Heat Treatment) Wheat Callus Transformed 64,1584,055,925 1.58% with MS26+ Homing Endonuclease (37 C. for 24 hours)

Identification of Mutations at the TaMS26 Target Site in RegeneratedPlants.

Red-fluorescing callus events were selected for plant regeneration.Plants were grown in the greenhouse and leaf DNA from individualregenerated wheat plants (n=122) was screened for MS26-1 target sitemutations by amplification of the region by PCR using the primer pairUNIMS26 5′-2 (GACGTGGTGCTCAACTTCGTGAT; SEQ ID NO: 26) and UNIMS26 3′-1(GCCATGGAGAGGATGGTCATCAT; SEQ ID NO: 27) and digestion of the amplifiedproducts with the DNA restriction enzyme, BsiWI, which recognizes thesequence 5′-CGTACG-3′. Products of these reactions were electrophoresedon 1% agarose gels and screened for BsiWI digestion resistant bandsindicative of mutations at the MS26-1 targets site.

Ten out of the 122 regenerated plants screened from PHP42063 heattreated embryos contained PCR products resistant to BsiWI restrictionenzyme digestion indicating mutations at the MS26 target site.Subcloning and DNA sequence analysis of these PCR products revealed avariety of mutations across the MS26 target site ranging from a singlenucleotide insertion to deletions of 4 to 98 nucleotides. In total,seven non-identical mutations were identified in these regenerated wheatplants (FIG. 3). Plants containing these mutations were allowed toself-pollinate. Progeny plants were screened for meiotic inheritance ofthe above describe mutations by PCR amplification to reveal BsiWIdigestion resistant bands indicative of mutations at the MS26-1 targetssite (as describe above). BsiWI resistant PCR amplification productswere identified in progeny plants grown from selfed seed derived parentplants which contained mutant Ms26 alleles, while DNA sequence analysisof these products confirmed sexual transmission of the original mutationto the next generation.

This example demonstrates the modification of wheat genes by directeddelivery of a double-strand-break reagent, in this case a custom homingendonuclease, which recognized, cleaved, and mutated wheat chromosomalDNA through end-joining repair.

That which is claimed:
 1. A construct comprising an expression cassette,wherein said expression cassette comprises a first promoter that drivesexpression of an operably-linked first polynucleotide in a wheat plant,wherein said first promoter is a male tissue-preferred promoterheterologous to the operably linked first polynucleotide, and whereinthe operably-linked first polynucleotide is an Ms26 polynucleotide thathas a nucleotide sequence selected from the group consisting of: (a) anucleotide sequence comprising SEQ ID NO: 7; (b) a nucleotide sequenceencoding a polypeptide comprising SEQ ID NO: 8, wherein said polypeptideincreases male fertility in a mutated male-sterile Ms26 wheat plant; (c)a nucleotide sequence comprising at least 90% sequence identity to thefull length sequence of SEQ ID NO: 7, wherein said nucleotide sequenceencodes a polypeptide that increases male fertility in a mutated Ms26male-sterile wheat plant; and (d) a nucleotide sequence encoding apolypeptide having at least 95% sequence identity to the full length ofthe polypeptide sequence of SEQ ID NO: 8, wherein said polypeptideincreases male fertility in a mutated Ms26 male-sterile wheat plant. 2.The construct of claim 1, further comprising a second polynucleotideoperably linked to a second promoter that drives expression in a plant,wherein the second promoter is heterologous to the operably-linkedsecond polynucleotide.
 3. The construct of claim 2, wherein said secondpolynucleotide encodes a gene product that interferes with male gametefunction, formation, or dispersal, wherein the gene product thatinterferes with male gamete function, formation, or dispersal encodesbarnase, DAM-methylase, amylase, or ADP ribosylase.
 4. The construct ofclaim 3, further comprising a third polynucleotide operably linked to athird promoter, wherein said third polynucleotide encodes a marker geneproduct, wherein the third promoter is heterologous to theoperably-linked third polynucleotide.
 5. The construct of claim 4,wherein said marker gene product comprises an antibiotic resistancemarker gene product or a visual marker gene product.
 6. A wheat plantcell comprising the construct of claim
 1. 7. A wheat plant comprisingthe plant cell of claim 6, wherein expression of said firstpolynucleotide increases the male fertility of said plant when comparedto a control plant.
 8. The wheat plant of claim 7, wherein theexpression of said first polynucleotide confers male fertility to a malesterile plant.
 9. A seed of the wheat plant of claim 7, wherein saidseed comprises said construct.
 10. A method of increasing male fertilityin a mutated Ms26 male sterile wheat plant, relative to a control plant,wherein said method comprises introducing into said wheat plant apolynucleotide operably linked to a male tissue-preferred promoterheterologous to the polynucleotide, wherein said promoter drivesexpression in the wheat plant, said polynucleotide comprising anucleotide sequence selected from the group consisting of: (a) anucleotide sequence comprising SEQ ID NO: 7; (b) a nucleotide sequenceencoding a polypeptide comprising SEQ ID NO: 8, wherein said polypeptideincreases male fertility in a mutated male-sterile Ms26 wheat plant; (c)a nucleotide sequence comprising at least 90% sequence identity to thefull length sequence of SEQ ID NO: 7, wherein said nucleotide sequenceencodes a polypeptide that increases male fertility in a mutated Ms26male-sterile wheat plant; and (d) a nucleotide sequence encoding apolypeptide having at least 95% sequence identity to the full length ofthe polypeptide sequence of SEQ ID NO: 8, wherein said polypeptideincreases male fertility in a mutated Ms26 male-sterile wheat plant, andwherein expression of the polynucleotide increases the wheat plant'smale fertility.
 11. The method of claim 10, wherein expression of saidpolynucleotide confers male fertility to a male sterile wheat plant. 12.The method of claim 10, wherein the polynucleotide that increases awheat plant's male fertility when expressed in the wheat plant isoperably linked to a polynucleotide encoding a marker gene product. 13.The method of claim 12, wherein the polynucleotide encoding a markergene product is an antibiotic resistance marker gene product or a visualmarker gene product.
 14. The method of claim 10, wherein thepolynucleotide that increases a wheat plant's male fertility whenexpressed the wheat plant is operably linked to a polynucleotideencoding a gene product that interferes with male gamete function,formation, or dispersal, wherein the gene product that interferes withmale gamete function, formation, or dispersal encodes barnase,DAM-methylase, amylase, or ADP ribosylase.